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Ancient Biomolecules, 2002 Vol. 4 (1), pp. 19–23 Species Determination using Species-discriminating PCRRFLP of Ancient DNA from Prehistoric Skeletal Remains J. BURGERa,b,*, R. SCHOONc, B. ZEIKEa, S. HUMMELa and B. HERRMANNa a Historical Anthropology and Human Ecology, University of Goettingen, Goettingen, Germany; bInstitute of Anthropology, University of Mainz, Mainz, Germany; cInstitute of Archaeology, Kiel, Germany (Received 12 September 2000; In final form 10 December 2000) Interspecific sequence polymorphisms in the mitochondrial cytochrome b gene were analyzed by PCR-RFLP to determine the species origin of Bronze Age animal skeletal remains. Existing techniques were refined by targeted primer design focusing on a DNA fragment shorter than 200 bp, an approach allowing us to identify up to six animal species at the same time. Possible contaminants, such as human DNA, were reliably ruled out. For routine applications in archaeometry, food or material analyses, PCR-RFLP may thus provide a simple alternative to sequencing of PCR products, allowing discrimination between species, even if the template DNA is degraded or contains traces of DNA from various species. costly procedure. We have therefore developed a timesaving and less expensive PCR-RFLP method (in the literature the term CAPS [cleaved amplified polymorphic site] is used synonymously to PCRRFLP), which allows reliable species identification from prehistoric animal bone samples, even those containing only degraded DNA. Keywords: Ancient DNA; Animal bone; CAPS; DNA extraction; DNA mixtures; PCR-RFLP; Species determination Figure 1 shows the result of amplification of modern DNA from seven different species with varying amounts of target DNA and varying annealing temperatures, using primers CB7u and CB7l. Template DNA from cattle, goat, and sheep, whose sequences have an almost complete match with the primers, was amplified under all conditions and produced strong bands. The differing amplification success for all other species can be explained by the alignment of the respective sequences with the two primers, as shown in Fig. 2. Within the 30 pentamer, the sequence of Cervus elaphus has no mismatch with CB7u (there are two mismatches outside the 30 pentamer), but there is one mismatch at the third position of CB7l (three outside). CB7u matches almost completely but CB7l does not, resulting in irregularities in the amplification. Although the C. elaphus specimen produced a band at 608C with 6.8 ng of DNA, there was no band at 608C and 34 ng of DNA. It is obvious that, under less stringent INTRODUCTION Determination of the species origin of (pre)historic objects is one of the common tasks of ancient DNA (aDNA) analysis. Usually, short fragments of conserved regions of mitochondrial DNA (mtDNA) are PCR amplified and then sequenced (e.g. Loreille et al., 1997). If a more detailed phylogenetic analysis of the obtained sequence is required or if the specimen contains extremely degraded DNA, the PCR products must be cloned before sequencing (Handt et al., 1996). However, for routine applications in archaeozoology or food analysis, this is a laborious and RESULTS Primer Characteristics and Species Discrimination *Corresponding author. Address: Institute of Anthropology, Johannes Gutenberg-University, SB II 02, D-55099 Mainz, Germany. Tel.: þ49-6131-39-2 4489. Fax: þ49-6131-39-2 5132. E-mail: [email protected] ISSN 1358-6122 print/ISSN 1607-8411 online q 2002 Taylor & Francis Ltd DOI: 10.1080/13586120290018491 20 J. BURGER et al. FIGURE 1 Amplification of 195 bp fragments of various species under varying conditions, using primers CB7u and CB7l. The annealing temperature and amount of target DNA is shown for each PCR. conditions (548C), although there is a mismatch with CB71 at the third position, C. elaphus template DNA was always amplified, though less efficiently. Within the 30 pentamer, the sequence of Capreolus capreolus shows one mismatch with CB7u at the second position (two outside) and one mismatch with CB7l at the third position (one outside). Under stringent conditions (608C), there was no amplification product visible for the roe deer. At 548C, a band appeared only with the larger amount of DNA target (34 ng), but even here, the amplification was not efficient. Within the 30 pentamer, the human cytochrome b sequence has two mismatches with CB7u at positions one and two (five outside) and one mismatch with CB7l at the third position (four outside). The 30 mismatch with CB7u and the numerous mismatches outside the 30 pentamer did not result in a significantly different amplification behavior compared with the roe deer. Homo sapiens, too, amplified only under non-stringent conditions and with a high copy number of targets, but less efficiently than roe deer DNA. FIGURE 3 Restriction profiles of the 195 bp cytochrome b PCR fragments obtained after treatment with Tsp509, showing interspecific polymorphism between O. aries, B. taurus, C. hircus, C. capreolus and C. elaphus. The ancient samples DoT 926 and DoT 904 show the fragment lengths of 105 and 77 bp characteristic for O. aries. DoT 1491 shows characteristic fragment lengths of 114 and 68 bp, as expected for B. taurus. DoT 1584a and DoT 1584b show a fragment of 182 bp, characteristic for C. hircus. The modern control of roe deer (C. capreolus, pos.) shows the expected band size of 162 bp, and deer (C. elaphus, pos.) shows the characteristic bands at 54 and 108 bp. ExA and ExB indicate two independent DNA extractions. Species Determination by PCR-RFLP The 195 bp PCR amplified fragment was digested with the restriction endonuclease Tsp509. Figure 3 shows the RFLP pattern for five of the specimens from the Lichtenstein Cave compared with various modern species as controls. Evidently, all species can be distinguished from one another and all specimens, including those derived from prehistoric remains, can be attributed to a given species: DoT 926 and DoT 904 show fragment lengths of 105 and 77 bp, characteristic for Ovis aries. (cf. Table II) DoT 1491 shows characteristic fragment lengths of 114 and 68 bp, as expected for Bos taurus. DoT 1584a and DoT 1584b each show a fragment of 182 bp, characteristic for Capra hircus. All results were obtained from two independent DNA extractions (ExA, ExB) and FIGURE 2 Alignment of the CB7u and CB7l primer annealing sequences for seven species (O. aries, sheep; O. aries musimon [subspecies], mouflon; C. hircus, goat; B. taurus, cattle; C. elaphus, deer; C. capreolus, roe deer, H. sapiens, human). SPECIES DETERMINATION BY PCR-RFLP 21 TABLE I Comparison of the results of morphological and genetic species determination Sample DoT 926 DoT 904 DoT 7 DoT 1491 DoT 1285 DoT 1584a DoT 1584b DoT 1584c DoT 1566 DoT 1468 DoT 1477 Morphological species Genetic species Ovis aries Ovis aries Ovis aries Bos taurus Bos taurus Capra hircus Capra hircus Capra hircus Capra hircus Capra hircus or Ovis aries Capra hircus or Ovis aries Ovis aries Ovis aries Ovis aries Bos taurus –* Capra hircus Capra hircus Capra hircus Capra hircus Capra hircus –* * No DNA available. confirmed several times by independent PCRs, as well as by DNA sequencing (results not shown). In Table I, the morphological species determination of all 11 specimens is compared with the results of the genetic analysis. Two of the examined bones did not contain ancient DNA. One previously unidentified bone (DoT 1468) was attributed to the species C. hircus. In all other cases, the species established by morphology was identical to the genetic species. Mixtures of Modern DNA In addition to the ancient DNA results, experiments with small amounts of mixed modern DNA were carried out. Figure 4 shows the RFLP pattern of mixtures with varying proportions of two to three species, and demonstrates that species can be identified specifically when template mixtures from two species are analyzed up to a mixture ratio of 59:1. Mixtures of three species are detectable as well. However, when minimizing target DNA, bands tend to fade away on the agarose gel. In general, shorter fragments are less intense than longer fragments, irrespective of the amount of target DNA. DISCUSSION Primer Design Human DNA is the most common source of contamination when working with ancient DNA. For this reason, amplification of human DNA should be avoided by use of maximally discriminating primers when working with animal material. The data presented here show that one mismatch within the 30 pentamer is not sufficient to exclude a sequence from amplification (see Fig. 1, C. elaphus ). However, one mismatch in each primer near the 30 end is sufficient to exclude a sequence either under stringent conditions or with little target in the reaction (see Fig. 1, C. capreolus ). Many additional FIGURE 4 Restriction cleavage patterns of DNA mixtures with varying proportions of target DNA. mismatches outside the 30 pentamer alter this situation only minimally (see Fig. 1, H. sapiens ). In the final analysis, the experiments show that, when using universal primers, it is necessary to carry out a sequence alignment with the desired species and to perform a PCR under varying conditions (temperature, target number). This is the only way to gain a more detailed knowledge about the annealing properties and thus amplification characteristics of primers, necessary when dealing with highly degraded material in general. Identifying the Species of Origin Our data clearly show that it is possible to identify the species of origin from circa 3000-year-old bone specimens by using RFLPs of a 195 bp PCR amplified fragment and at the same time to exclude possible (human) contamination. If the locus and the restriction enzyme are chosen properly, this procedure can be applied to other species or populations as well. Certainly, the principle of PCR-RFLP is not new and is widely used, for example in the field of food analysis (e.g. Meyer et al., 1995; Plath et al., 1997; Carrera et al., 2000). However, fulfilling each of the four criteria mentioned in Materials and Methods (see below) (presence of a RFLP specific for the considered species, fragment length of less than 200 bp, exclusion of contaminating sequences, and a restriction site within at least one of the primers) optimizes and refines existing methods, making them applicable to analysis of truly old and highly degraded DNA, but at the same time makes the experimental design slightly more laborious. In difficult cases, it is possible to combine several enzymes and thus to accumulate restriction sites (data not shown). Here, enzymes should be added one at a time to the PCR product, not simultaneously. 22 J. BURGER et al. Using PCR-RFLP analysis, we were additionally able to identify DNA from up to three species within template mixtures. This result is of particular interest when foodstuffs containing mixtures of components are to be analyzed. The same is true for the examination of (pre)historic artifacts, such as glues, binders, consumer goods, or food incrustations in containers, which usually consist of several components. Figure 4 shows the possibility of identifying mixtures, even in proportions of 59:1. Note here that the detection limit can certainly be improved by using PAGE instead of an agarose gel. This might also reduce the observed phenomenon of shading of shorter bands on agarose gels. Reliable analysis of DNA mixtures may also be valuable when frequent contaminants, such as cattle, cannot be excluded by discriminative primer design. While direct sequencing of coamplified endogenous DNA and contaminant DNA would lead to multiple sequences, PCRRFLP is able to separate the two signals. Thus, PCRRFLP is a rapid alternative method of species determination from highly degraded DNA, even when multiple organisms contribute to a given sample, and can be applied when the exact sequence information of a specimen is not required. MATERIALS AND METHODS Lichtenstein Cave in Osterode, Harz, Germany, is a Bronze Age archeological site dating to 900– 700 BC. In addition to numerous human skeletons, the cave contains bones from some autochthonous animal species. Earlier analysis showed that the state of DNA preservation in the bones is excellent, mainly due to the low temperature prevailing in the cave since prehistoric times (Burger et al., 1999). Eleven animal bone samples were chosen for aDNA analysis. With a high degree of certainty, three of them were morphologically attributed to the species Ovis aries (sheep), two to Bos taurus (cattle) and four to C. hircus (goat). In two cases, the bone fragments of sheep and goat could not clearly be distinguished. All samples underwent the standard procedures for aDNA work as previously described (Burger et al., 2000). water (Ampuwa, Fresenius) was added. As the extraction procedure was automated the volumes of reagents dispensed may have varied between runs. Five hundred microliters of Proteinase K was added and the mixture incubated for 1 h at 588C with shaking. Three milliliters of phenol/chloroform/isoamyl alcohol (25:24:1, pH 7.5 – 8.0) was added and the mixture was further incubated at room temperature for 6 min with shaking. The phases were allowed to separate by incubating at room temperature for 8 min without shaking and the organic phase and interphase, if present, were discarded. Chloroform (4.5 ml, 100%) was added to the aqueous phase and the mixture incubated for 6 min at room temperature while shaking. The phases were again allowed to separate by incubating at room temperature for 8 min without shaking and the organic phase was discarded. Ninety microliters of sodium acetate (pH 4.5) and 3.2 ml of 100% isopropanol were added followed by incubation for 2 min with shaking. Five microliters of Glasmilk (Dianova) was added and the suspension was shaken for another 10 min. To obtain a pellet, the solution was filtered through Precipitette filters (Applied Biosystems) or centrifuged for 3 min at 5000 rpm. The pellet was washed with 80% ethanol and eluted into 50 ml sterile distilled water (Ampuwa, Fresenius). Five to ten microliters of extract were used for PCR amplification or the extract was stored at 2208C. Glasmilk was not removed prior to amplification. Primer Design Primers were designed to fulfill the following criteria (see also Fig. 2): primers ought to have a maximum match with the sequences of the targeted animal species, in this case sheep, goat, and cattle; primers ought to have a maximum mismatch with the corresponding human sequence; primers should amplify a sequence of less than 200 bp; the sequence amplified should contain a recognizable RFLP between sheep, cattle, goat, and possible other species, here deer and roe deer; and at least one primer should contain a restriction site serving as a reaction control. DNA Extraction PCR Tooth or bone samples were roughly ground with a pestle and mortar, then finely powdered in a Retsch mill. Bone/tooth powder (0.3 g) was incubated in 1.5 ml of 0.5 M EDTA (pH 8.3) for 20 h while rotating. The suspension was centrifuged for 4 min at 4000 rpm. The supernatant was transferred to a fresh tube or to an automated nucleic acids extraction system (Nucleic Acid Extractor 341A, Applied Biosystems) and 1.6 ml sterile distilled A 195 bp segment of the mitochondrial cytochrome b gene was amplified using the primers CB7u: 50 GCGTACGCAATCTTACGATCAA-30 and CB7l: 50 CTGGCCTCCAATTCATGTGAG-30 . The PCRs were carried out in 50 ml of 60 mM KCl; 12 mM Tris –HCl; 2.5 mM MgCl2; 150 mM dNTPs; 0.18 mM each primer; and 2 U AmpliTaq Gold (PE Applied Biosystems). The temperature profile was 948C for 1 min, 54 or 608C for 1 min, and 728C for 1 min, for 32 cycles. SPECIES DETERMINATION BY PCR-RFLP TABLE II Fragment lengths for various species after digestion of the 195 bp PCR product with restriction enzyme Tsp509 Species Fragment lengths (bp) Ovis aries (sheep) Capra hircus (goat) Bos taurus (cattle) Cervus elaphus (deer) Capreolus capreolus (roe deer) Homo sapiens (human) 13, 75, 105 13, 182 3, 68, 114 13, 20, 54, 108 13, 20, 162 12, 182 Known amounts of modern DNA (3.4, 6.8, 34, 68 ng) were amplified at 54 and at 608C. Five microliters of ancient DNA extracts from the bones was used for PCR amplification without separation of the nucleic acids from the Glasmilk. Modern DNA in mixtures of various proportions (1:1 to 59:1 and 1:1:1) consisting of two to three species (sheep, goat, cattle) were also used in PCRs. Restriction Endonuclease Digestion Seven microliters of PCR product, 12 U restriction enzyme Tsp509 (New England Biolabs) and 2 ml buffer (New England Biolabs) were incubated for 2 h at 658C. Fragments were separated in 2.5% agarose gels for 2 h. Expected fragment lengths are shown in Table II. 23 Acknowledgments We thank Stephan Flindt for providing samples and Helen Fletcher and Ekkehard May for assistance. We thank the German Ministry for Education and Research for financial support. References Burger, J., Hummel, S., Herrmann, B. and Henke, W. (1999) “DNA preservation: a microsatellite-DNA study on ancient skeletal remains”, Electrophoresis 20, 1722–1728. Burger, J., Hummel, S., Pfeiffer, I. and Herrmann, B. (2000) “Palaeogenetic analysis of (pre)historic artifacts and its significance for anthropology”, Anthropol. Anz. 58, 69–76. Carrera, E., Garcia, T., Cespedes, A., Gonzalez, I., Fernandez, A., Asensio, L.M., Hernandez, P.E. and Martin, R. (2000) “Identification of smoked Atlantic salmon (Salmo salar ) and rainbow trout (Oncorhynchus mykiss ) using PCR-restriction fragment length polymorphism of the p53 gene”, J.A.O.A.C. Int. 83, 341–346. Handt, O., Krings, M., Ward, R.H. and Pääbo, S. (1996) “The retrieval of ancient human DNA sequences”, Am. J. Hum. Genet. 59, 368–376. Loreille, O., Vigne, J.D., Hardy, C., Callou, C., Treinen-Claustre, F., Dennebouy, N. and Monnerot, M. (1997) “First distinction of sheep and goat archaeological bones by the means of their fossil mtDNA”, J. Archaeol. Sci. 24, 33–37. Meyer, R., Höfelein, C., Lüthy, J. and Candrian, U. (1995) “Polymerase chain reaction-restriction fragment length polymorphisms analysis: a simple method for species identification in food”, J.A.O.A.C. Int. 78(6), 1542–1551. Plath, A., Krause, I. and Einspannier, R. (1997) “Species identification in dairy products by three different DNAbased techniques”, Z. Lebensm. Unters. Forsch. A. 205, 437 –441.